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An AFM Study of Lipid Monolayers. 2. Effect of Cholesterol on Fatty Acids E. Sparr,*,† K. Ekelund,‡ J. Engblom,‡ S. Engstro¨m,‡ and H. Wennerstro¨m† Department of Physical Chemistry 1, Chemical Center, Lund University, Lund, Sweden, and Department of Food Technology, Chemical Center, Lund University, Lund, Sweden Received January 29, 1999. In Final Form: May 28, 1999 In this study the effect of cholesterol in Langmuir-Blodgett monolayers of fatty acids of varying chain lengths was investigated by atomic force microscopy (AFM). Domain formation due to lateral phase separation was studied at different lipid compositions and surface pressures. A small amount of cholesterol is miscible with palmitic acid (C16:0) and forms a flat monolayer while excess cholesterol forms a rougher cholesterolrich phase. No miscibility was observed in monolayers of lignoceric acid (C24:0) and cholesterol. For the ternary mixed monolayer (palmitic acid, lignoceric acid, and cholesterol) the two fatty acids formed separate domains and the miscibility of cholesterol in the two phases showed behavior corresponding to that of the binary fatty acid-cholesterol systems. From the shape, size, and height differences of the domains one can conclude that the driving force to minimize the interfacial length between different phases is reduced in the presence of cholesterol. This can be attributed to line active properties of cholesterol.
Introduction Langmuir-Blodgett (LB) monolayers can be used as simplified models for biological membranes. It is welldocumented that membrane constituents are not always homogeneously arranged in the membrane bilayers but rather organized in lateral microdomains. Two-dimensional phase separations and phase transitions in LB films have therefore been intensively studied. Classically, the phase behavior of surfactant mixtures at the gas-liquid interface has been characterized by means of surface pressure-area isotherms.1,2 Fluorescence microscopy,3 Brewster-angle microscopy,4 and X-ray diffraction5 have also been used to study lateral arrangement of lipids at the interface, although none of these techniques can provide information on heterogeneous domain formation as well as on nanometer scale structures. AFM is a surface imaging technique with very high lateral resolution. It can be used for nondestructive investigations of monolayer structures on solid supports. There is a general belief that the structure of a monolayer at the air-water interface is simply correlated to the structure of the monolayer transferred to a solid substrate,6 and several studies on molecular arrangement of LB lipid films by AFM has been carried out. In particular AFM has been exploited to investigate the detailed structure of phase-separated LB films,7 as well as lateral forces (friction)8 and adhesion forces.9 In this paper we extend a previous study of domain formation in fatty acid monolayers10 and investigate the effect of cholesterol, which is an important constituent in many membranes not least those in the outermost layer * Corresponding author. † Department of Physical Chemistry. ‡ Department of Food Technology. (1) Sta¨llberg-Stenhagen, S.; Stenhagen, E. Nature 1945, 3956, 239240. (2) Do¨rfler, H. D.; Koth, C. Colloid Polym. Sci. 1992, 270, 384-391. (3) Subramaniam, S.; McConnell, H. M. J. Phys. Chem. 1987, 91, 1715-1718. (4) Wolthaus, L.; Schaper, A.; Mo¨bius, D. J. Phys. Chem. 1994, 98, 10809-10813. (5) Kenn, R. M.; Bo¨hm, C.; Bibo, A. M.; Peterson, I. R.; Mo¨hwald, H.; Als-Nielsen, J.; Kjaer, K. J. Phys. Chem. 1991, 95, 2095-2097. (6) Schwartz, D. K.; Viswanathan, R.; Garnaes, J.; Zasadzinski, J. A. J. Am. Chem. Soc. 1993, 115, 7374-7380.
of skin, stratum corneum (SC). Its molecular structure implies unique functions such as to stabilize and fluidize lipid mono-and bilayers.11,12 Cholesterol also influences the lateral domain formation, acting as a “lineactant” (cf. surfactant) in the boundary region between two-dimensional gel and liquid crystalline phases.13 Lipids in biological membranes are generally in a liquid crystalline state, which corresponds to an expanded state of the monolayer, although there are a few exceptions. The extracellular lipid of stratum corneum mainly consists of lipids in a crystalline (gel) state.14 These crystalline domains has been envisioned as a mosaic held together by lipids in a liquid crystalline state.15 It has been found that drugs and water are mainly transported through this lamellar matrix16 and consequently the phase behavior of the lipids is of great importance for the understanding of the barrier function of skin. The major lipids present in SC are ceramides, cholesterol, and free fatty acids of different chain lengths and degrees of saturation.17,18 It is our opinion that a deeper understanding of the effect of cholesterol on individual skin lipids and their mixtures is fundamental for the understanding of phase behavior, domain formation, and barrier properties of SC lipids. Here we present an AFM study on topographical and frictional properties of mixed fatty acids and cholesterol monolayers. Focus is on the domain formation and how it is effected by cholesterol, lipid miscibility, and surface (7) ten Grotenhuis, E.; Demel, R. A.; Ponec, M.; Boer, D. R.; van Miltenburg, J. C.; Bouwstra, J. A. Biophys. J. 1996, 71, 1489-1399. (8) Overney, R. M.; Meyer, E.; Frommer, J.; Gu¨ntherodt, H.-J. Langmuir 1994, 10, 1281-1286. (9) Dufreˆne, Y. F.; Barger, W. R.; Green, J.-B. D.; Lee, G. U. Langmuir 1997, 13, 4779-4784. (10) Ekelund, K.; Sparr, E.; Engblom, J.; Engstro¨m, S.; Wennerstro¨m, H. Langmuir 1999, 15, 6946. (11) Vist, M. R.; Davis, J. H. Biochemistry 1990, 29, 451-464. (12) Smaby, J. M.; Brockman, H. L. Langmuir 1992, 8, 563-570. (13) McConnell, H. M.; Keller, D.; Gaub, H. J. Phys. Chem. 1986, 90, 1717-1721. (14) Bouwstra, J. A.; Gooris, G. S.; Salomons-de Vries, M. A.; van der Spek, J. A.; Bras, W. Int. J. Pharm. 1992, 84, 205-216. (15) Forslind, B. Acta Derm.-Venereol. 1994, 74, 1-6. (16) Elias, P. M. J. Invest. Derm. Suppl. 1983, 80, 44-49. (17) Wertz, P. W.; Swartzendruber, D. C.; Madison, K. C.; Downing, D. T. J. Invest. Dermatol. 1987, 89, 419-425. (18) Norle´n, L.; Nicander, I.; Lundh Rozell, R.; Ollmar, S.; Forslind, B. J. Invest. Dermatol. 1999, 112, 72-77.
10.1021/la9900932 CCC: $15.00 © 1999 American Chemical Society Published on Web 08/05/1999
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pressure. This work is preceded by a thorough investigation on monolayers of single free fatty acids of different chain lengths and their mixtures.10 By the combination of results from monolayer studies and X-ray diffraction measurements of bulk lipids,19 a closer insight into membrane lipid organization can be achieved. Materials and Methods Palmitic acid (C15H31COOH) and lignoceric acid (C23H47COOH) (+99% purity) were purchased from Larodan Fine Chemicals (Malmo¨, Sweden) and cholesterol (+99% purity) was from Sigma Chemicals (St. Louis, MO). Monolayers were prepared on a Langmuir-Blodgett trough type 611 from Nima Technology (Coventry, England). A 0.1 M acetate buffer, adjusted to pH 4.0, was used as subphase. The water was deionized, distilled and filtered through a Millipore Q purification system (Millipore Corp., Bedford, MA). Fatty acids and cholesterol were dissolved in chloroform (1 mg/mL) and spread at the air-water interface. Isotherms were reversible and reproducible. For deposition, sheets of freshly cleaved mica were immersed into the subphase. After the chloroform was allowed to evaporate for 20 min, the monolayer film was compressed at a speed of 20 cm2/min to the pressure of deposition. The monolayer was left standing at constant pressure for 20 min before it was transferred to the substrate with a dipping speed of 2 mm/min. All samples were prepared in a cleanroom at a constant temperature of 19 °C. The transfer ratios of the monolayers were close to unity. Constant force AFM and lateral force AFM (LFM)20 measurements were performed on a commercial Nanoscope IIIa instrument (Digital Instruments, Santa Barbara, CA). Experiments were run under an air atmosphere at ambient temperature within 4 h from sample preparation. An E tube scanner with a 10 × 10 (x, y) × 2.5 (z) µm scan range was used for imaging. Microfabricated square pyramidal shaped tips of silicon nitride with a bending spring constant of 0.12 N/m (manufacture specified, Digital Instruments, Santa Barbara, CA) were used as received. The scan rate was 2 Hz, and the applied force was in the order of 1-10 nN. To eliminate imaging artifacts, the scan direction was varied to ensure a true image. Images were obtained from at least five macroscopically separated areas on each sample. All images were processed using procedures for plane-fit and flatten in Nanoscope IIIa software version 4.22 (Digital Instruments, Santa Barbara, CA) without any filtering. Dimensions of the domains were measured directly from the AFM height images, and thickness variations were estimated from section analysis of the topographic images.
Results Surface Pressure-Area Isotherms. The surface pressure-area isotherms of the systems were carefully studied before depositing the monolayers on the mica support. Surface pressure-area isotherms were recorded for monolayers of lignoceric acid (C24:0) and palmitic acid (C16:0) mixed with cholesterol. We have previously reported surface pressure-area isotherms of lignoceric acid and palmitic acid.10 Lignoceric acid showed a firstorder phase transition between an liquid expanded and a liquid condensed film around 8-10 mN/m and 22-25 Å2/molecule, palmitic acid showed a continuous phase transition around 22 mN/m and 22 Å2/molecule, and the equimolar mixture gave an isotherm where the phase transitions of both components were present, indicating immiscibility of these fatty acids. The shape of the lignoceric acid isotherm does not change significantly upon addition of cholesterol. Addition of a small amount of cholesterol to palmitic acid (1:0.01 molar fraction of fatty acid-cholesterol) results in a less pronounced kink of the palmitic acid isotherm that disappears gradually on (19) Engblom, J.; Engstro¨m, S.; Jo¨nsson, B. J. Controlled Release 1998, 52, 271-280. (20) Neubauer, G.; Cohen, S. R.; McClelland, G. M.; Horne, D.; Mate, C. M. Rev. Sci. Instrum. 1990, 61, 2296-2308.
Figure 1. Surface pressure-area isotherm for monolayers of lignoceric acid-palmitic acid-cholesterol, molar ratio 1:1:x, where x ) 010-0.4. Deposition pressures used for the experiments are indicated in the isotherms (A-D).
Figure 2. Topographic AFM image (3 × 3 µm) of a transferred monolayer of lignoceric acid-cholesterol molar ratio 1:0.01. Small domains of cholesterol are embedded in a monolayer of lignoceric acid. The height difference between the phases was measured to 1.5 nm. The film was deposited on mica at a surface pressure corresponding to C in Figure 1. Z range: 6 nm.
increasing cholesterol content. At equimolar mixture of palmitic acid and cholesterol the monolayer exhibits a surface pressure-area behavior analogous to that of pure cholesterol. When cholesterol is added to this mixture of fatty acids, a less pronounced phase transition for the palmitic acid is observed as for the palmitic acidcholesterol case, while the transition of lignoceric acid is unchanged (Figure 1). The transition from gaseous to liquid expanded state of the monolayers was clearly observed but not studied in detail. Binary Fatty Acid-Cholesterol Monolayers. AFM images of a transferred monolayer of lignoceric acid and cholesterol, molar ratio 1:0.01, were monitored for a range of surface pressures representing the liquid expanded state to the liquid condensed state of lignoceric acid monolayers. In all samples we observe small circular domains of cholesterol (appear dark in Figure 2) of a typical diameter of 50-100 nm, embedded in a matrix of lignoceric acid. The circular domains are 1.5 nm thinner than the surrounding phase, which is consistent with the expected height difference between monolayers of lignoceric acid and cholesterol. An extended hydrocarbon chain in a monolayer of lignoceric acid should correspond to a total length of 3.1 nm, and the stiff cholesterol molecule has a total length of about 1.6 nm. Area ratios between the two different phases show a good agreement with the composition of the sample, taking into account the different headgroup areas of the two species. Taken together, these results indicate that cholesterol does not mix with the lignoceric acid monolayer of a liquid expanded or liquid condensed state. This conclusion is also supported by the surface pressure-area isotherm (Figure 1), where no
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Figure 3. Topographic AFM images (3 × 3 µm) of a transferred monolayer of palmitic acid-cholesterol. (a) Molar ratio 1:0.01. The film is homogeneous, indicating miscibility of the lipids. Z range: 3 nm. (b) Molar ratio 1:0.05. Two phases can be observed, one “large” flat phase (referred to as I) and one rough phase (referred to as II). Z range: 3 nm. (c) Molar ratio 1:0.4. Small flat “flakes” (I) are embedded in a lower rough (II) phase. The height difference between the phases was measured to 0.4 nm. Z range: 4 nm. The flat (I) phase in (a)-(c) is assumed to be palmitic acid with a small amount of cholesterol, while the rougher (II) phase in (b) and (c) is assumed to be a cholesterol-rich phase. The films were deposited on mica at a surface pressure corresponding to D in Figure 1.
influence on the phase transition was observed when adding cholesterol. In the transferred monolayer of lignoceric acid and cholesterol, molar ratio 1:0.4 (image not shown), the cholesterol forms a continuous phase, embedding “islands” of lignoceric acid. The shapes of the domains were irregular but with a smooth perimeter. The height (Figure 3a) and friction images of a transferred monolayer of palmitic acid and cholesterol in 1:0.01 molar mixture were homogeneous and flat, and no domains of cholesterol were observed. The same result was found for a monolayers at surface pressures corresponding to both liquid expanded and liquid condensed states (image not shown). From this, one can conclude that palmitic acid interacts with a small amount of cholesterol to such an extent that they are miscible. Furthermore, the palmitic acid film was flatter after addition of cholesterol. The cholesterol-containing film also seemed to be more robust and less damaged by the tip than the pure fatty acid film. The cholesterol molecule consists of one rigid steroid skeleton with low conformational freedom and a flexible (isooctyl) chain. The molecular properties can facilitate close packing of hydrocarbon chains without causing crystallization. On further addition of cholesterol the lipids are no longer miscible. A monolayer of palmitic acid and cholesterol in molar ratio 1:0.05 shows two phases where the thinner phase is more rough and exhibits higher friction (Figure 3b). With even higher cholesterol content (molar ratio 1:0.4) (Figure 3c) the difference between the two phases is more distinct. A thicker flat phase forms flakes in the thinner rough phase. The height differences between the two phases was measured to 0.4 nm, which is in good agreement with the predicted difference between the palmitic acid monolayer (2.0 nm) and cholesterol (1.6 nm). It was also observed that the area ratio of the flat phase decreased with increasing amount of cholesterol. These results show the onset of a phase separation in the mixed monolayer for palmitic acid-cholesterol molar ratios between 1:0.01 and 1:0.05. The monolayer height differences indicate that the thicker phase consists mainly of palmitic acid and the thinner phase is rich in cholesterol. Experiments with small amounts of cholesterol in palmitic acid have shown miscibility (Figure 3a), leading to the conclusion that higher flat phases in Figure 3b,c consist of a closely packed monolayer of palmitic acid and a small amount of cholesterol. From area ratios one can further conclude that the thinner phase must include both cholesterol and palmitic acid. When the surface pressure is increased, the flakes of the thicker phase grow and form bigger domains, although the height difference and the area ratio between the two phases do not change significantly.
Figure 4. AFM images (3 × 3 µm) of a transferred monolayer of palmitic acid-lignoceric acid-cholesterol. (a) Topographic image for molar ratio 1:1:0.01. Two phases can be observed, one thick lignoceric acid phase (referred to as III) and one thinner phase (I; see Figure 3). The height difference between III and I was measured to 1.1 nm. (b) Topographic image (top) and friction image (bottom) for molar ratio 1:1:0.2. Three phases can be observed. The thicker phase consists of lignoceric acid (III), and the thinner phases are mixed monolayers of palmitic acid and cholesterol (I and II). Height differences were measured to 1.1 nm for phases I-III and 1.5 nm for phases II-III. The difference between the two low phases is clearly seen in the friction image, as well as the coexistence of lignoceric acid liquid condensed and liquid expanded phases, as described in ref 10. (c) Topographic image for molar ratio 1:1:2. Two phases can be observed, one thick phase (III) and one thin phase (II). Height difference between III and II was measured to 1.5 nm. The films were deposited on mica at a surface pressure corresponding to B (b) and D (a, c) in Figure 1. Z range: for the topographic image, 5 nm; for the friction image, 0.1 V.
Mixed Fatty Acids and Cholesterol Ternary Monolayers. We have previously shown that lignoceric acid and palmitic acid monolayers are not miscible and form domains with a height difference of 1.1 nm.10 Addition of cholesterol to an equimolar mixture of these fatty acids shows a behavior which is consistent with the results of the binary fatty acid-cholesterol mixtures. For small amounts of cholesterol two phases are observed where a higher phase of lignoceric acid is embedded in a continuous phase of palmitic acid and cholesterol (Figure 4a). For higher amounts of cholesterol a third phase appears as illustrated by the monolayer of lignoceric acid, palmitic
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acid, and cholesterol at a molar ratio of 1:1:0.2 (Figure 4b). In this image the thicker phase (appears bright in the Figure 4b top image) is the condensed monolayer of lignoceric acid. It is consistent with the observations on the binary systems that the thinner rough phase is a cholesterol-rich mixed monolayer of palmitic acid and cholesterol, while the intermediate phase consists of a monolayer of palmitic acid with a small amount of cholesterol, corresponding to the “flakes” in Figure 3c. The latter phase is mainly found along the boarder of the lignoceric acid phase. For high cholesterol concentrations this phase also appears as small “flakes” in the thinner cholesterol-rich phase. All three phases are observed at surface pressures ranging from 10 to 22 mN/m. When the surface pressure is increased, the small flakes disappear and the palmitic acid-rich phase is exclusively found along the boundary toward the lignoceric acid monolayer. The palmitic acid domains are of long and irregular shapes with nonminimized interfaces toward the surrounding cholesterol-rich phase, as was also seen in the palmitic acid-cholesterol monolayers (Figure 3c). The height differences between the lignoceric acid domains and the thinner phases were measured to 1.1 and 1.5 nm, respectively, supporting the assignment of the palmitic acid-rich and cholesterol-rich phases. Monolayers of the equimolar mixture of the fatty acids and excess cholesterol (molar ratio 1:1:2) show two phases (Figure 4c) with a height difference of 1.5 nm, corresponding to a thicker lignoceric acid monolayer and a thinner monolayer of palmitic acid and cholesterol referred to as a cholesterolrich phase. Discussion Phase Behavior. The surface pressure-area isotherms and the direct AFM observations give a consistent picture of the phase behavior in the monolayer and show that palmitic acid and cholesterol form a homogeneous monolayer at cholesterol content ratios lower than 1:0.05. With increasing cholesterol concentration, a cholesterol-rich monolayer exists in equilibrium with a palmitic acid-rich monolayer. At even higher cholesterol concentration only one phase is observed which corresponds to the cholesterolrich phase. The exact compositions of the phase boundaries were not investigated in this work. Similar phase behaviors have also been reported for mixed phospholipidcholesterol monolayers at surface pressures below a miscibility critical point.21,22 Motomura et al.23 have studied mixed monolayers of cholesterol and fatty acids. On the basis of surface pressure-area isotherms and thermodynamic considerations, these authors suggests total miscibility between myristic acid (C14:0) and a low fraction of cholesterol at low surface pressures, while they propose a phase separation into a monolayer of pure cholesterol in equilibrium with an expanded two-component cholesterol-poor monolayer for high cholesterol concentrations. Total immiscibility of the lipids at surface pressures corresponding to a condensed state of the fatty acid monolayer was also claimed. These conclusions do not fully agree with our results for the transferred monolayers of palmitic acid and cholesterol. We suggest an equilibrium between two mixed monolayers of different compositions rather than the formation of a pure cholesterol phase in monolayers of low cholesterol content. (21) Hagen, J. P.; McConnell, H. M. Biochim. Biophys. Acta 1997, 1329, 7-11. (22) Slotte, J. P. Biochim. Biophys. Acta Biomembr. 1995, 1235, 419427. (23) Motomura, K.; Terazono, T.; Matuo, H.; Matuura, R. J. Colloid Interface Sci. 1976, 57, 52-7.
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Additionally, we observe this phase behavior both at intermediate and at high surface pressures, and there is no separation into two one-component phases for surface pressures corresponding to the liquid condensed phase of the pure fatty acid. The difference between our observations and those of Motomura et al. can be due to the two carbon difference in the chain of the fatty acid. We observe that a small amount of cholesterol makes the transition kink less pronounced in the isotherm for the palmitic acid. This indicates that cholesterol reduces the cooperativity of the continuous liquid expanded to liquid condensed transition in the film. Although this can be the effect of any impurity, cholesterol has some rather unique properties relative to the packing of saturated fatty acid chains. It allows for a dense packing of straight chains without requiring long-range crystalline order.11 This interpretation is consistent with our observation that in the AFM measurements there are no visible differences in the monolayers for surface pressures corresponding to the liquid expanded and the liquid condensed state of the palmitic acid monolayer. There is always a possibility that the monolayer structure is changed during the deposition procedure. A very small variation of monolayer area during the transfer may cause unwanted phase transitions of monolayers exhibiting a continuous phase transition. However, the pressure-area curves and the AFM observations are systematically consistent and the described phase behavior of palmitic acid and cholesterol applies for the whole range of surface pressures from 10 to 22 mN/m. Thus, we believe that the observed phase behavior for mixed palmitic acid cholesterol monolayers reflects the behavior at the air-water interface. AFM studies on mixed monolayers of cholesterol, ceramides, and fatty acids at high surface pressures have been presented by ten Grotenhuis et al.7 Their results show solubility of palmitic acid in a phase of short-chained ceramides and cholesterol, although some small “features” in the film were reported. This may support our conclusion that palmitic acid is miscible in a cholesterol-rich phase to some extent, and the “features” can then be seen as small domains of palmitic acid with a minor amount of cholesterol. Lignoceric acid is not miscible with either palmitic acid or cholesterol to any observable extent. The association of cholesterol with lipids in a monolayer is mainly determined by the acyl chain length. Experiments on phospholipids of different chain lengths have shown the best “match” for cholesterol and saturated acyl chains having 14-17 carbons.21,24 This interaction can be explained from steric packing constraints of the hydrocarbon chains, due to the character of the cholesterol molecule. The same mechanism should apply to the interaction between cholesterol and the acyl chain of free fatty acids explaining the difference in miscibility of cholesterol within the fatty acid monolayers. Domain Formation. The AFM images of the mixed monolayers show small domains within the lignoceric acid phase. These domains are also observed in the monolayer of lignoceric acid and palmitic acid without cholesterol.10 For samples with low cholesterol content, these should have the same composition as the large continuous phase of palmitic acid and cholesterol, showing the same height differences compared to the lignoceric acid phase. In the samples with higher cholesterol content, the small domains show an inhomogeneous film structure, and one can assume coexistence of the cholesterol rich and the palmitic acid-rich phases. This interpretation is consistent (24) Slotte, J. P. Biochim. Biophys. Acta 1995, 1238, 118-126.
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Figure 5. Histogram showing the variation in interfacial length with cholesterol content. The relative increase in interfacial length is measured for the small domains within the lignoceric acid phase.
with the observed height differences between the phases. With increasing cholesterol content, the number of small domains increases, while their total area remains unchanged (i.e. smaller domains), resulting in an increased interfacial length between the condensed lignoceric acid monolayer and the more loosely packed cholesterol-rich phase. Figure 5 illustrates the dependence of interfacial length of these small domains on cholesterol concentration. As a consequence of its unique chemical structure, the cholesterol molecule locates preferentially at the twodimensional solid-liquid interface, leading to a reduced line tension along the interface. Cholesterol can therefore be regarded as a line active (cf. surface active) agent between two-dimensional liquid crystalline and gel phases. A theoretical treatment of the line active properties of cholesterol was given by Keller et al.25 The line activity of cholesterol was also demonstrated in epifluorescence microscopy studies on monolayers of various phospholipids at the air-water interface.13,26 In these studies, the domains have a typical size of a few micrometers, which is significantly larger than the domains reported in this work. The line active effect of cholesterol evidently influences the monolayer arrangement on various length scales. Computer simulations on cholesterol-phospholipid bulk behavior have also indicated cholesterol to act as an interfacial active agent that prefers to be located in the boundaries between gel and fluid domains of the bilayer.27 It can be noticed that the shape of the lignoceric acid domains does not change on addition of cholesterol, indicating that these domains are kinetically trapped structures. To further examine this aspect, a monolayer (lignoceric acid, palmitic acid, and cholesterol, molar ratio 1:1:0.1) was left at the pressure of 0 mN/m for 12 h before depositing at 22 mN/m (Figure 6a). In the previous study a 1:1 molar mixture of lignoceric and palmitic acid was prepared in similar way (Figure 6b).10 In the cholesterolcontaining sample, the lignoceric acid domains are of an irregular shape, although the interfaces between the two phases are smoother than for the samples that have not been equilibrated at zero pressure for 12 h. The number (25) Keller, D. J.; McConnell, H. M.; Moy, V. T. J. Phys. Chem. 1986, 90, 2311-2315. (26) Weis, R. M.; McConnell, H. M. J. Phys. Chem. 1985, 89, 44534459. (27) Cruzeiro-Hansson, L.; Ipsen, J. H.; Mouritsen, O. G. Biochim. Biophys. Acta 1989, 979, 166-176.
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Figure 6. Topographic AFM image (8 × 8 µm) of the transferred monolayers of (a) palmitic acid-lignoceric acid-cholesterol, molar ratio 1:1:0.01, and (b) palmitic acid-lignoceric acid, molar ratio 1:1, from ref 10. The films were relaxing at a surface pressure of 0 mN/m for 12 h before deposited on mica at a surface pressure of 22 mN/m. Z range: 5 nm.
of small domains in the lignoceric acid phase is roughly the same as for the earlier described cholesterol-containing samples. This result is significantly different from the cholesterol-free sample which showed very regular shaped domains of lignoceric acid of uniform size and few small “lakes” of palmitic acid.10 From these results, it emerges that the presence of cholesterol reduces the driving force for minimizing the interface between the lignoceric acid and palmitic acid monolayers. Cholesterol thus decreases the interfacial line tension between the two phases and can also here be seen as a line active agent in this system. The majority of the studies undertaken to elucidate the effects of cholesterol on lipid morphology in bulk have been performed on systems containing various phospholipids, due to their frequent occurrence in biological membranes. Cholesterol is soluble with phospholipids in the crystalline (gel) state to some extent. It has been referred as a “crystal breaker” as it disturbs the translational order of the phospholipid molecules.11,28 Cholesterol also causes a straightening of the disordered phospholipid acyl chains in the liquid crystalline phase and reduces the mean headgroup area.11 This property is often referred to as the stabilizing effect of cholesterol. Similar effects of cholesterol on free fatty acids in liquid crystalline and gel phases can be expected. Results obtained by Engblom et al.19 using SWAXD (small- and wide-angle X-ray diffraction) on free fatty acid-cholesterol mixtures correlate well with the above findings. Here the free fatty acids used were palmitic and oleic acid, neutralized to 41 mol %. Cholesterol showed a preference for the unsaturated oleic acid, independently of the palmitic acid:oleic acid ratios. A liquid crystalline phase was evident at low cholesterol concentrations that increased with increasing amount oleic acid. This phase became more gel-like with additional cholesterol, and at 33 mol % cholesterol a separate crystalline cholesterol phase was observed. The results of the present study indicate that effect of cholesterol on fatty acids monolayers (28) Ipsen, J. H.; Karlstro¨m, G.; Mouritsen, O. G.; Wennerstro¨m, H.; Zuckermann, M. J. Biochim. Biophys. Acta 1987, 905, 162-172.
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also shows a correlation with the phase behavior of the bulk lipid bilayers. Conclusion Cholesterol is immiscible in lignoceric acid monolayer but miscible to a small degree in palmitic acid monolayers, where in addition a cholesterol-rich phase incorporates substantial amounts of palmitic acid. This reflects that there is a good matching between the bulky cholesterol molecule and the shorter hydrocarbon chain of the palmitic acid. At higher cholesterol concentrations there are coexisting phases in the mixed palmitic acid-cholesterol monolayer with a flat palmitic acid-rich phase and a rough
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cholesterol-rich phase. An effect of cholesterol in monolayer domain formation is the reduced line tension along the boundaries between the condensed and expanded phases. This is visualized by an increased interfacial length per domain where cholesterol can be regarded as a line active agent between the liquid condensed lignoceric acid domains and the more expanded palmitic acid/cholesterol phase. Acknowledgment. Liselotte Eriksson is gratefully acknowledged for providing Figure 4c. LA9900932
